Microscopic examination of tissue samples, particularly those obtained by biopsy, is a common method for diagnosis of disease. In particular, immunohistochemistry (IHC), a technique in which specific antibodies are used to detect expression of specific proteins in the tissue sample, is a valuable tool for diagnosis, particularly for the detection and diagnosis of cancer.
Lung cancer is the leading cause of cancer death for both men and women. More people die of lung cancer than of colon, breast, and prostate cancers combined. Non-small cell lung carcinoma (NSCLC) comprises approximately 80% of lung cancers and may be classified into several histological types, most commonly include adenocarcinoma (ADC) or even squamous cell carcinoma (SCC). Classification of lung carcinomas into histological types may be performed by morphological examination using hematoxylin and eosin (H&E) or immunohistochemistry, and in some cases even, mucin stains; however, accurate classification can be difficult with poorly differentiated or even undifferentiated lung carcinoma. Diagnosis can be further complicated by the use of needle core biopsies, which provide limited amounts of tissue for both immunohistochemistry and molecular testing, and may include crush artifacts. Additionally, cytology specimens may lack morphological features necessary for diagnosis with H&E alone.
Although the majority of lung cancers (particularly grades I and II) can be diagnosed with only H&E staining, with the advent of targeted therapies, diagnostic needs have changed, and an improved method for classification of a greater number of NSCLC cases is needed. In the past, histologic subtyping of NSCLCs had limited diagnostic value, due to the fact that the same treatment may have been provided to the patient, perhaps regardless of NSCLC subtype. However, the availability of targeted therapies has created a need for accurate subtyping of NSCLC. For example, bevacizumab, a therapeutic humanized monoclonal antibody targeting vascular endothelial growth factor, may be a common treatment for NSCLC patients; however, patients with the SCC subtype should not receive bevacizumab, perhaps due to the about 30% mortality rate by fatal pulmonary hemorrhage. Furthermore, enhanced efficacy may have been demonstrated with the addition of premextred to conventional chemotherapy in non-squamous cell carcinomas, but may not in SCC. Therefore, accurate methods for subtyping NSCLC specimens may be useful for the best patient care, with optimal therapeutic efficacy and minimal adverse effects.
Immunohistochemistry may be commonly used to assist pathologists in determining histologic subtype of NSCLC specimens, perhaps particularly discriminating ADC from SCC, as well as from Small Cell Carcinomas of the lung. Historically, the antibodies TTF-1 and p63 may have been used in IHC to differentiate primary adenocarcinoma from squamous cell carcinoma of the lung. Both of these antibodies may suffer from limitations in sensitivity (e.g. TTF-1 may stain only about 70% of ADC cases) or specificity (p63, a marker for SCC, may stain about 11% of ADC cases). In order to improve sensitivity and specificity, antibody combinations may have been suggested for use in a panel that may improve diagnostic accuracy for histologic subtyping, perhaps over use of a single antibody. For example, in one study, a panel of TTF-1, p63, Napsin A and even CK5/6 was used to classify about 77% of poorly differentiated cases of NSCLC; however about 23% of the cases remained unclassified. Similarly, a five antibody panel that may include CK5/6, TRIM29, LAT-1, CEACAM5 and even MUC1 perhaps may be used in a weighted mathematical formula to classify about 85% of LADC cases and about 88% of lung SCC cases, respectively, perhaps while leaving about 12.8% of the cases unclassified. IHC with Napsin A and p63 may have also been suggested as a method to discriminate ADC from SCC. Each of these antibody panels may face certain limitations or deficiencies. For example, in each of these methods, multiple sections of a specimen may need to be stained, in order to obtain the diagnosis; this may be undesirable because limited tissue may be available and it may need to be conserved for other testing. Additionally, each of these methods may be unable to provide a diagnosis for all cases, leaving some specimens unclassified (e.g. limited sensitivity or the like), or they may inaccurately identify a histologic subtype (e.g. limited specificity or the like).
In one example, a series of cocktails containing two or more (e.g., at least two) primary antibodies may have been used in a two-color staining IHC procedure (also known as a “double-stain” or “Multiplex”) perhaps to classify specimens as ADC or SCC in a more efficient manner and may even use fewer sections of the tissue specimen. When used in a diagnostic sequence, cocktails of Napsin A+Desmoglein-3, TTF-1+CK-5, and p63+TRIM-29, were about 94.7% sensitive and about 100% specific for ADC and SCC, in this study. With this method, about 7.1% of the specimens remained unclassified.
Several antibodies are known to be used independently, which may be useful in IHC methods and which may even provide increased sensitivity, specificity, and/or classification percentage, consume less of a specimen for testing, or even exhibit other advantages or the like.
Desmoglein 3 (DSG3 or DSG-3) may be a calcium-binding transmembrane glycoprotein component of desmosomes in vertebrate epithelial cells. As a result, IHC using a DSG3 antibody may produce membranous staining, perhaps not cytoplasmic. In one study, DSG3 was reported to stain about 98% of cases of SCC, while about 99% of non-SCC cases were negative.
Napsin A, a novel aspartic proteinase, may be normally expressed in type II pneumocytes, alveolar macrophages, renal tubules, exocrine glands, and even pancreatic ducts. Studies have shown that Napsin A may be a very specific marker for lung adenocarcinoma. The role of Napsin A in differentiating primary from metastatic ACA of the lung may have been reported. Although, it may occasionally stain non-pulmonary ACAs, Napsin A may be a useful marker in differentiating primary lung ACAs from SCCs. Positive immunohistochemical staining with Napsin A may show intense granular cytoplasmic reactivity.
Thyroid transcription factor-1 (TTF-1) may be a member of the NKX2 family of homeodomain transcription factors and may result in nuclear specific staining in IHC. It may be expressed in epithelial cells of the thyroid gland and even the lung. In one study, TTF-1 may have had a sensitivity of about 70% for cases of ADC, with a specificity of about 94.7% versus SCC (about 5.3% of SCC cases stained with TTF-1).
p63 may be a member of the p53 family of transcription factors. In normal tissues, IHC with p63 may be positive in squamous epithelia, in basal cells of urothelium and even in basal cells of prostate epithelium. IHC using a p63 antibody may result in nuclear staining. p63 may be detected in greater than about 80% of lung squamous cell carcinomas; however, greater than about 10% of lung adenocarcinomas may also stain for p63, which may be a limitation of specificity that may result in an equivocal or perhaps even an inaccurate diagnosis.
p40 is an isoform of the p63 gene family that may lack the N-terminal transactivation domain of p63. In IHC using a p40 antibody, one study found equal sensitivity for SCC when using p40 or p63; however, p40 may have exhibited superior specificity. In this study, p63 stained about 31% of lung ADC cases, but p40 stained only about 3% of these cases. IHC using a p40 antibody may result in nuclear staining. p40 and p63 may exhibit similar staining patterns and may be interchangeable for certain applications.
Cytokeratins (CK) may be the dominant, intermediate filament proteins of the epithelial cells. CK5 may be detected in normal cells, including: breast myoepithelial cells, prostate basal cells, and perhaps even the basal layer of the epidermis and even salivary glands. Positive immunohistochemical staining of CK5 may display a cytoplasmic staining pattern, which may be indicative of SCC. Positive immunohistochemical staining of CK7 may display a cytoplasmic staining pattern, which may be indicative of lung ADC.
Given the current state of the art, IHC methods with increased sensitivity and/or specificity in the classification of histologic subtypes of NSCLC specimens may be useful. Similarly, methods that have potential to classify a greater percentage of specimens (e.g., fewer specimens remain unclassified with the method), or even consume less of a specimen for testing, may also be useful. Additionally, methods that may improve agreement in classification between pathologists (e.g., concordance) may be valuable. Also, methods that may assist in determining the origin of a tumor, particularly as from the lung or an organ other than the lung, may be useful.